Natural and Medical Sciences Institut at the University of Tübingen
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1 NMI is alliance partner of Baden-Württemberg Innovation Alliance Dr. Udo Kraushaar Head Electrophysiology Bringing human neuronal biology to HTS: Functional Drug Screening with icell GlutaNeurons and Astrocyte on the Hamamatsu FDSS/µCELL Natural and Medical Sciences Institut at the University of Tübingen
2 NMI Natural and Medical Sciences Institute at the University Tübingen Established as a foundation under public law (non-profit organization) in employees Highly interdisciplinary (Physics, Biophysics, Molecular Biology, Biochemistry, Cell Biology) Applied R&D, and services for industrial clients Joint projects with academic and industrial partners Incubator site >17 spin-off companies founded Pharmaservices CRO offering pharmaservice
3 Cell Models / Technology At EPhys Lab Cell- and Organ Systems Core site Factsforkids.com CNS SC derived cells Cardio elsitemaendocrinoupel.blogspot.com Pancreas Freeimages.com ENS Methods & Assay Development Retina Public funding Industrial customers Life-cell imaging MEA Patch clamp 3 Technology
4 CDI GlutaNeurons on Hamamatsu µcell 1) Establishment of stable assay, 384 well format - Cell density - Cultivation procedure & time window - Buffers & dyes - Getting to know the expected activity patterns 2) icell GlutaNeurons vs. Cocultivation with Astrocytes + Morphology 3) Functional applications - Seizurogenic compound tests (HESI NeuTox subset) - Network dynamics by mglur agonists - (simple) disease model Single-cell patch clamp MEA network activity 4
5 Basics: Buffer, Dye, Cell density, Coating Recording buffer 2.5Ca 2+ /0.2Mg 2+ vs 2.5Ca 2+ /0Mg 2+ Ca 2+ indicator Cal520 vs Fluo4 30 sec Cell density 30k/well vs 20k/well Coating (21 DIV) PLO/Matrigel vs 0.1%PEI 5 At time of first experiments lack of ready-to-use protocol First suggestions
6 Basics: Buffer, Dye, Cell density, Coating Recording buffer 2.5Ca 2+ /0.2Mg 2+ vs 2.5Ca 2+ /0Mg 2+ Ca 2+ indicator Cal520 vs Fluo4 30 sec Cell density 30k/well vs 20k/well Coating (21 DIV) PLO/Matrigel vs 0.1%PEI 6 Coating: PLO/Matrigel 30k cells/well (384 well format) Cal520-AM (45 min, 2 µm, 37 C, Pluronic) Recording Buffer 2.5 mm Ca 2+ /0.2 mm Mg 2+, HEPES Stable recording time window DIV14-21
7 Network activity is +/- independent on cocultivation icell GlutaNeurons icell GlutaNeurons icell Astrocytes + icell Astrocytes 100 sec Activity Amplitude Rise Area 7
8 Seizurogenic compounds in vitro surrogate for epilepsy Compound MoA Concentration 4-Aminopyridine (4-AP) K + channel blocker (IK A ) 1-30 µm Picrotoxin GABA A channel blocker µm Pentylenetetrazole (PTZ) GABA A / K + channel blocker (?) µm Strychnine Glycin/ACh channel blocker 1-30 µm Pilocarpine Muscarinergic ACh agonist 1-30 µm 5 out of 12 HESI NeuTox compounds 8-10/90% rise time - Peak amplitude - Area under the curve Investigated parameters: - Activity rate - CV of signal regularity
9 Pentylenetetrazol (PTZ) (GABA A, K + blocker) PTZ 9 30 sec Ctrl Increased area Increased amplitude Increased activity
10 4-Aminopyridine (IK A blocker) Normalized Activity Increased area Increased amplitude Increased activity 10
11 Picrotoxin (GABA A blocker) Normalized Activity Increased area Increased amplitude Increased activity 11
12 Seizurogenic compounds Compound MoA Concentration 4-Aminopyridine (4-AP) K + channel blocker (IK A ) 1-30 µm Picrotoxin GABA A channel blocker µm Pentylenetetrazole (PTZ) GABA A / K + channel blocker (?) µm Strychnine Glycin/ACh channel blocker 1-30 µm Pilocarpine Muscarinergic ACh agonist 1-30 µm Concentration-dependent seizure-like network activity Sensitivity equal or higher compared to primary neurons Cocultivation with Astrocytes more robustness 12
13 mglur agonists mglurs: G-protein coupled receptors, modulation of synaptic transmission/excitability Compound MoA Expected action DHPG LY mglur I/V agonist GABA A AMPA mglur II agonist Glu release Seizurogenic Network activity reduction L-AP4 mglur III agonist Network activity reduction Picrotoxin + DHPG Pilocarpin + L-AP4 Increased seizures, prolonged discharge Partial recovery from seizure 13
14 DHPG (mglur I agonist, seizurogenic) Increased area Increased activity seizure-like activity 14
15 LY (mglur II agonist, network activity reduction) GlutaNeurons failed after C1 Speed up risetime Reduced activity 15
16 L-AP4 (mglur III agonist, network activity reduction) Increased area Speed up risetime Massively reduced activity 16
17 L-AP4 (network activity reduction) 5 min post application 20 min post application norm. Beats per Minute norm. Beats per Minute 30 sec 17
18 L-AP4 disturbs network synchronicity, not spike rate control E1 E2.... L-AP4 E10 24-well MEA Single well Electrode layout 100 s 18 Microelectrode array (MEA), icell GlutaNeurons
19 DHPG fails to increase seizurogenic activity in the presence of Picrotoxin Protocol: - Control recording - Preincubation 20 min Picrotoxin - Application of DHPG Amplitude Area Activity Preliminary data: n=3 19
20 L-AP4 partially recovers seizurogenic effect of Pilocarpine Protocol: - Control recording - Preincubation 20 min Pilocarpine - Application of L-AP4 Amplitude Area Activity norm. Amplitude norm. Area norm. Activity Preliminary data: n=3 20
21 Amyloid-beta42 application as simple Alzheimer model - Neurotoxic main component of amyloid plaques - Main candidate to form misfolded version of tau - Active form a Uptake by neurons - Experiment: - treatment 24 h prior to experiment - Data normalized to DMSO controls Wikipedia.de 21
22 Amyloid-beta42 reduces network activity concentrationdependently 22
23 Summary Hamamatsu µcell allows to record strong and stable network activity of icell Glutaneurons 384-well format makes experiments fast and cell-saving (=cheap) Seizurogenic compounds and mglur agonists modulate network activity of icell Glutaneurons concentration-dependently Similar or higher sensitivity compared to primary neurons Neurotox assays Neuromodulation research (Alzheimer, Parkinson, Schizophrenia) Cocultivation with icell Astrocytes Does not alter base network parameters Allow more reproducable recordings compared to icell GlutaNeurons alone Allow faster recovery from mglur III activation (L-AP4, to be further investigated) Pathological effects by Amyloid- reproducible Disease modelling 23
24 Acknowledgement NMI: Electrophysiology: Sandra Buckenmaier Karin Gebhardt Dominik Loser (MEA data) Molecular Neurobiology: Martin Kriebel (primary neurons, A ) Cellular Dynamics: Cell sponsoring Sabine Lange Blake Anson Hamamatsu: Organization of Meeting Novartis Pharma: Hosting of Meeting 24
25 Contact Dr. Udo Kraushaar Head Electrophysiology NMI Natural and Medical Sciences Institute at the University of Tuebingen Markwiesenstr Reutlingen Germany Phone:
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